Borrelia burgdorferi and related spirochetes are invasive, nontoxigenic pathogens that cause the long-term infection and multiple manifestations associated with Lyme borreliosis. Until recently, genetic manipulations of low passage B. burgdorferi with retention of infectivity have not been possible. Presence of the linear plasmids Ip25 and Ip28-1 correlates with infectivity, and recent studies have shown that Ip25 and another linear plasmid, Ip56, greatly reduce the transformation rates of Borrelia; rare clones that have spontaneously lost Ip25 (and thus infectivity) are preferentially transformed by shuttle vectors. Preliminary data presented in this application confirms that the large open reading frame BBE02 in Ip25 encodes an enzyme with both restriction and modification activities. A disruption mutant of BBE02 has been isolated that is readily transformable with a shuttle plasmid and retains infectivity. In addition, a recent publication by our group demonstrates that introduction of the Ip25 gene BBE22 (pncA) restores infectivity to noninfectious clones lacking Ip25. Taken together, these findings provide the genetic tools required for a systematic analysis of the virulence determinants of B. burgdorferi, as described in this proposal. In Specific Aim 1, site-directed mutagenesis will be used to disrupt a number of genes thought to be involved in the mammalian or tick phases of the Borrelia life cycle, including ospAB, ospC, dbpAB, ospEF, and visE. The effects of these mutations on infection of C3H/HeN mice and ticks will be investigated, and the correlation with gene function confirmed by gene complementation studies. The goal of Specific Aim 2 is to identify additional genes that are required in the infectious cycle. In vitro transposon mutagenesis or alternatively signature-tagged mutagenesis will be utilized to randomly disrupt genes in an infectious, highly transformable B. burgdorferi B31 clone lacking the Ip25 and Ip56 restriction-modification systems that interfere with transformation. These approaches will, for the first time, permit a systematic, global analysis of virulence determinants of Lyme disease Borrelia and will thus provide insight into the mechanisms of pathogenesis of these highly invasive spirochetes. [unreadable] [unreadable]